The present invention relates to a method for producing an oligosaccharide. The present invention also relates to a novel oligosaccharide and a pharmaceutical composition containing it.
Keratan sulfate is a glycosaminoglycan comprising, as a basic structure, N-acetyl lactosamine in which the 6-position of the N-acetyl glucosamine residue is O-sulfated. It has been reported that some of degradation products of karatan sulfate, i.e., keratan sulfate oligosaccharides, have a pharmacological activity (see, for example, International Publication No. WO96/16973).
As a disaccharide structure derived from keratan sulfate, those shown in FIG. 1 may be expected. However, searching of another keratan sulfate oligosaccharide having a pharmacological activity has been limited, because it has been difficult to obtain a keratan sulfate oligosaccharide as a disaccharide having a galactose residue at its reducing end (GlcNAcxcex21xe2x86x923Gal where Gal represents galactose, GlcN represents glucosamine, and Ac represents an acetyl group) and having a sulfate group like keratan sulfate. For example, even by treating keratan sulfate with known endo-xcex2-galactosidases, it is difficult to obtain a disaccharide having a galactose residue at its reducing end (GlcNAcxcex21xe2x86x923Gal) with a sulfate group retained.
A first object of the present invention is to provide a method that makes it possible to easily produce a keratan sulfate disaccharide having a galactose residue at its reducing end.
A second object of the present invention is to provide a novel oligosaccharide having a pharmacological activity, and to provide it as a medicine.
The inventors of the present invention found that a disaccharide having a galactose residue at its reducing end could be obtained through glycosidic linkage formation by protecting hydroxyl groups and an amino group in glucosamine, and hydroxyl groups in galactose in a particular manner. Moreover, they also found that disaccharides in which hydroxyl groups at particular positions were sulfated had excellent pharmacological activities. The present invention has been accomplished based on these findings.
The present invention provides a method for producing an oligolsaccharide represented by the following general formula (3): 
wherein R10 and R11 each independently represent a hydrogen atom or xe2x80x94SO3M where M represents a proton or a monovalent cation, Ac represents an acetyl group, R12 represents a hydrogen atom, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue, or a peptide residue, and Z represents an oxygen atom or xe2x80x94NHCOxe2x80x94,
which method comprises at least a step of carrying out glycosidic linkage formation between a monosaccharide represented by the following general formula (1): 
xe2x80x83wherein R1 and R2 each independently represent an aralkyl group, R3 represents an acyl group or a silyl group, R4 represents a protective group for an amino group, and R5 represents a leaving group, and
a monosaccharide represented by the following general formula (2): 
xe2x80x83wherein R6 and R8 each independently represent an aralkyl group, R7 represents an acyl group or a silyl group, R9 represents an aralkyl group, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue, or a peptide residue, and Z represents an oxygen atom or xe2x80x94NHCOxe2x80x94 (this method is also referred to as the xe2x80x9cproduction method of the present inventionxe2x80x9d hereinafter).
In the production method of the present invention, it is preferred that at least one of R10 and R11 represents xe2x80x94SO3M where M represents a proton or a monovalent cation, and the method comprises, after the step of carrying out the glycosidic linkage formation between the monosaccharide represented by the aforementioned general formula (1) and the monosaccharide represented by the aforementioned general formula (2), a step of substituting a hydrogen atom for at least one of R3 and R7, and subsequently substituting xe2x80x94SO3M for the hydrogen atom.
In a preferred embodiment of the production method of the present invention, the aforementioned general formulae (1)-(3) are represented by the following formulae (4)-(6), respectively: 
wherein Bn represents a benzyl group, R13 represents an acetyl group or a levulinoyl group, and Phth represents a phthaloyl group, and X represents a halogen atom; 
wherein Bn represents a benzyl group, R14 represents a benzyl group, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue, or a peptide residue, Z represents an oxygen atom or xe2x80x94NHCOxe2x80x94, and Piv represents a pivaloyl group; and 
wherein, Ac represents acetyl group, and M represents a proton or a monovalent cation, R15 represents a hydrogen atom, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue, or a peptide residue, and Z represents an oxygen atom or xe2x80x94NHCOxe2x80x94.
In another preferred embodiment of the production method of the present invention, the aforementioned general formulae (1)-(3) are represented by the following formulae (7)-(9), respectively: 
wherein, Bn represents a benzyl group, Lev represents a levulinoyl group, and Phth represents a phthaloyl group, and X represents a halogen atom; 
wherein, Bn, R14 and Z are as defined above, Ph represents a phenyl group, and Me represents a methyl group; and 
wherein, R15 and Z are as defined above, Ac represents an acetyl group, and M represents a proton or a monovalent cation.
In a further preferred embodiment of the production method of the present invention, the aforementioned general formulae (1)-(3) are represented by the following formulae (10)-(12), respectively: 
wherein, Bn represents a benzyl group, Lev represents a levulinoyl group, and Phth represents a phthaloyl group, and X represents a halogen atom; 
wherein, Bn, R14 and Z are as defined above, Ph represents a phenyl group, and Me represents a methyl group; and 
wherein, R15 and Z are as defined above, Ac represents an acetyl group, and M represents a proton or a monovalent cation.
The present invention also provides an oligosaccharide represented by the following general formula (13): 
wherein, R16 and R17 each independently represent a hydrogen atom or xe2x80x94SO3M where M represents a proton or a monovalent cation, Ac represents an acetyl group, R18 represents a hydrogen atom, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue, or a peptide residue, and Z represents an oxygen atom or xe2x80x94NHCOxe2x80x94, provided that an oligosaccharide wherein both of R16 and R17 are hydrogen atoms, Z is an oxygen atom and R18 is a hydrogen atom or a cholestanyl group, and an oligosaccharide wherein R16 represents xe2x80x94SO3M where M represents a proton or a monovalent cation, Z is an oxygen atom and both of R17 and R18 are hydrogen atoms are excluded (The oligosaccharide is also referred to as the xe2x80x9coligosaccharide of the present inventionxe2x80x9d hereinafter).
In a preferred embodiment of the oligosaccharide of the present invention, each of R16 and R17 is xe2x80x94SO3M where M represents a proton or a monovalent cation.
In another preferred embodiment of the oligosaccharide of the present invention, R16 is a hydrogen atom, and R17 is xe2x80x94SO3M where M represents a proton or a monovalent cation.
In the oligosaccharide of the present invention, R18 is preferably a hydrogen atom, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, an O-alkylglycerol residue or a cholestanyl group, and Z is an oxygen atom.
The present invention further provides a medicine comprising, as an active ingredient, the oligosaccharide of the present invention represented by the following general formula (13): 
wherein, R16 and R17 each independently represent a hydrogen atom or xe2x80x94SO3M where M represents a proton or a monovalent cation, Ac represents an acetyl group, R18 represents a hydrogen atom, a 6-O-sulfated N-acetylglucosamine residue, an alkyl group, a glycerol residue, an O-alkylglycerol residue, an O-acylglycerol residue, a cholesterol residue, a cholestanyl group, a ceramide residue, a phospholipid residue, a biotin residue, or a peptide residue, and Z represents an oxygen atom or xe2x80x94NHCOxe2x80x94, provided that an oligosaccharide wherein both of R16 and R17 are hydrogen atoms, Z is an oxygen atom and R18 is a hydrogen atom or a cholestanyl group is excluded, or a pharmaceutically acceptable salt thereof (The medicine is also referred to as the xe2x80x9cmedicine of the present inventionxe2x80x9d hereinafter).
In particular, the oligosaccharide of the present invention wherein R16 is xe2x80x94SO3M, and R17 is a hydrogen atom or xe2x80x94SO3M where M represents a proton or a monovalent cation, or a pharmaceutically acceptable salt thereof is useful as an anti-allergy agent, and the oligosaccharide of the present invention wherein both of R16 and R17 are xe2x80x94SO3M where M represents a proton or a monovalent cation, or a pharmaceutically acceptable salt thereof is useful as an anti-inflammatory agent.
The present invention also provides a pharmaceutical composition comprising the oligosaccharide of the present invention usable for the medicine and a pharmaceutically acceptable carrier. In particular, an anti-allergy composition comprising the oligosaccharide of the present invention usable for the anti-allergy agent and a pharmaceutically acceptable carrier, and an anti-inflammatory composition comprising the oligosaccharide of the present invention usable for the anti-inflammatory agent and a pharmaceutically acceptable carrier are provided. In addition, the present invention provides a method for preventing or treating allergy, which comprises administering a therapeutically effective amount of the oligosaccharide of the present invention usable for the anti-allergy agent to a subject in need of such treatment, and a method for preventing or treating inflammation, which comprises administering a therapeutically effective amount of the oligosaccharide of the present invention usable for the anti-inflammatory agent to a subject in need of such treatment.